title: Detection of islet β-cell death in vivo by multiplex PCR analysis of differentially methylated DNA.
abstract: Noninvasive detection of early β-cell death in type 1 diabetes might identify individuals in whom therapeutic interventions would preserve β-cell mass and prevent hyperglycemia. Recent studies in mice have shown that β-cell death produces a corresponding increase in unmethylated preproinsulin (PPI) DNA in serum. Here, we report the development of a novel assay using dual fluorescent-probe multiplex PCR (TaqMan) to detect differential methylation of circulating PPI DNA. Key assay features include low background signals, linear assay output across a large range of values, and simultaneous detection of methylated and unmethylated PPI DNA in a single reaction. We defined the "unmethylation index" as a summary parameter that reflects the relative amounts of unmethylated vs methylated PPI DNA. To validate this assay's ability to detect β-cell death in vivo, we measured the unmethylation index in the serum of diabetic mouse models, including high- and multiple low-dose streptozotocin-induced diabetes, and the nonobese diabetic mouse model of type 1 diabetes. Our data show a significantly increased unmethylation index concordant with the known timeline of β-cell death that precedes the onset of hyperglycemia. Subsequently, we observed a decrease in the unmethylation index following diabetes development, likely reflecting the absence of further β-cell death in the pancreas. We conclude that simultaneous measurement of methylated and unmethylated PPI DNA using the multiplex PCR method described here is a readily available and sensitive indicator of dying β-cells that may be useful to track diabetes progression and response to therapeutic intervention.
authors.affiliation: Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
authors.forename: Marisa M
authors.initials: MM
authors.lastname: Fisher
authors.forename: Cristina N
authors.initials: CN
authors.lastname: Perez Chumbiauca
authors.forename: Kieren J
authors.initials: KJ
authors.lastname: Mather
authors.forename: Raghavendra G
authors.initials: RG
authors.lastname: Mirmira
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Tersey
mesh_terms: D000465:Algorithms; D000818:Animals; D017209:Apoptosis; D016923:Cell Death; D004247:DNA / Q000097:blood*; D019175:DNA Methylation; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000175:diagnosis* / Q000473:pathology; D004195:Disease Models, Animal; D005260:Female; D007328:Insulin / Q000097:blood* / Q000235:genetics; D050417:Insulin-Secreting Cells / Q000473:pathology*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D016513:Mice, SCID; D060885:Multiplex Polymerase Chain Reaction; D009336:Necrosis; D011498:Protein Precursors / Q000097:blood* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013047:Specific Pathogen-Free Organisms
chemical_list: D007328:Insulin; D011498:Protein Precursors; C013737:preproinsulin; D004247:DNA